PROJECTSUMMARY: Inheritedcongenitalanteriorsegmentdysgenesis(ASD)disordersareamajorcauseofvision impairmentandblindness.Theyincludedevelopmentaloculardeficienciesoftheiris,corneaandlens inadditiontosignificantpredisposition,~50%,toglaucoma.Fromvariousstudiesinrecentyearsit hasbecomeclearthatmanycongenitaldisordersandpredispositionsarisefromearlydevelopmental defects,bothmajorandsubtle.Examiningthedevelopmentalprocessgivingrisetotheseessential componentsofthevisualsystemisthereforeafruitfulapproachtostudyingthedisease.Theanterior segmentderiveslargelyfromtheearlymigratingneuralcrestcellsthattargetspecificallytothe anteriorofthedevelopingretinaandarelooselyclassifiedasperiocularmesenchyme(POM).POM deficiencyhasbeendemonstratedincertaincasesofASD.Ibelieveamajorroadblockineffortsto treatandunderstandASDhasbeenthelimitedexaminationofPOMbiologyandfunctionduringearly oculardevelopment.Thishamperseffortstocharacterizetheetiologyofdiseaseandscreeningin ASDfamilies,inadditiontodelayingattemptsatiPSC-mediatedapproachesfortreatmentofASD.To circumventtheseproblems,weaimtocomprehensivelycharacterizePOMcellspecification, migration,targeting,differentiation,andultimatelycontributiontoanteriorsegmentdysgenesis.Thisis thenextlogicalandnecessarystepinorderforthefieldtocontinuetheevolutionoftherapyand screeningforglaucomaandASDdisorders.OurimmediategoalistotestthehypothesisthatPOM cellsdelineateintovarioussubpopulationsandspecifytobecometheanteriorsegment mesenchyme(ASM).Totestourhypothesisweproposetoemploythehighlyversatilezebrafish embryomodelsystemandcomprehensivelyexaminePOMcelldevelopmentwhilecatalogingtheir contributiontotheanteriorsegmentthroughgenetic,molecularandcuttingedgeinvivoimaging approaches.Ourspecificaimsare:Aim1:Characterizespecificationanddefinethecompositionof theASMtheusingacombinationofgeneexpressionanalysispairedwithlight-sheetinvivotime lapseimagingandconfocalmicroscopy.Aim2:Trackthemigratorybehaviorandlineagetrace distinctpopulationsofASMduringanteriorsegmentformationusinglight-sheetinvivotimelapse imagingandconfocalmicroscopypairedwithanovelfatemappingoptogenetictracingmechanism. Aim3:GenerateacomprehensiveASMtranscriptomeforscreeningnoveltargetsinASDpatient samplesusingacombinationofflowcytometrycoupledtoRNAseqandnextgenerationexome sequencing.